Interactions between the Nicotinic and Endocannabinoid Receptors at the Plasma Membrane

Compartmentalization, together with transbilayer and lateral asymmetries, provide the structural foundation for functional specializations at the cell surface, including the active role of the lipid microenvironment in the modulation of membrane-bound proteins. The chemical synapse, the site where neurotransmitter-coded signals are decoded by neurotransmitter receptors, adds another layer of complexity to the plasma membrane architectural intricacy, mainly due to the need to accommodate a sizeable number of molecules in a minute subcellular compartment with dimensions barely reaching the micrometer. In this review, we discuss how nature has developed suitable adjustments to accommodate different types of membrane-bound receptors and scaffolding proteins via membrane microdomains, and how this “effort-sharing” mechanism has evolved to optimize crosstalk, separation, or coupling, where/when appropriate. We focus on a fast ligand-gated neurotransmitter receptor, the nicotinic acetylcholine receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as a paradigmatic example.

Introduction
Current studies on biological membranes support the notion that they constitute highly specialized structures with compartmentalized functional regions of varying thickness, lipid composition, and functional properties. Signaling pathways require a highly regulated membrane organization since these processes are characteristically dynamic and occur in separated spatio-temporal discrete domains. Of great relevance to cell physiology are the lateral membrane heterogeneities enriched in cholesterol, sphingolipids, and phospholipids with saturated fatty acid acyl chains. Their chemical composition determines their engrossed thickness and their physicochemical properties are akin to those of the liquid-ordered (Lo) domains observed in artificial membranes. These membrane regions, also known as lipid rafts [1,2], play a crucial role in manifold cellular processes by providing the structural substrate for functional compartmentalization. Distinct cellular signaling mechanisms, in particular those operating at the cell-surface membrane, often rely on these discrete lateral domains to perform in an isolated manner in close but separate locations of the membrane. Interactions between domains harboring different signaling proteins can result in the dynamic regulation or modulation of these signaling mechanisms in their synergic potentiation, or their silencing. One example of such an interacting signaling system is discussed in this short review: the endocannabinoid (EC) system and the nicotinic acetylcholine receptors (nAChRs).
The EC system comprises a wide variety of lipid-signaling molecules, enzymes, and receptors. Endocannabinoids (ECs) are important homeostatic modulators of neuronal activity in the central nervous system (CNS). nAChRs are a Cys-loop gene family of neurotransmitter receptors, belonging to the superfamily of pentameric ligand-gated cation Resident proteins and interacting lipids within the plasma membrane dynamically orchestrate many signaling cascades. Alterations of the membrane lipid composition can result in defective functions of raft-associated proteins, and consequently favor abnormal cell signaling. For example, abnormal cholesterol metabolism has been implicated in the development of neurological disorders such as Alzheimer and Parkinson diseases, Huntington disease, Niemann-Pick type C disease, and schizophrenia spectrum disorders [28][29][30][31][32]. Likewise, alteration of membrane-associated proteins can also hamper many important cell functions and even lead to pathological states. In Alzheimer and Parkinson diseases, loss of plasma membrane integrity in neuronal cells is often observed. This condition favors toxic amyloid-β and α-synuclein aggregation [33][34][35] through lipid-induced conformational changes [34,[36][37][38] or mass action [39,40] that, when combined, increase the probability of inter-molecular interactions and promote aggregation [39,40]. During aggregation, amyloid-β oligomers extract phospholipids from the plasma membrane and incorporate these lipid molecules into the growing fibrils, thus causing membrane rupture. Disease proteins can increase membrane permeability by different mechanisms [40], such as protein-induced membrane rigidity [41,42], membrane thinning [43,44] and deformation [43][44][45][46], as well as detergent-like effects [38,41,47], and pore formation [36,37,42,[48][49][50]. These pores in the bilayer result in Ca 2+ influx from the extracellular compartment and efflux of cytosolic content.
Many proteins at the synapse dynamically modulate synaptic activity, thus influencing a great variety of biochemical states and protein-protein interactions that control protein synthesis and contribute to the reorganization of cytoskeletal architecture [51], which is essential for adequate dendritic spine morphology [52]. For instance, actin filaments can associate with actin-binding proteins (ABPs) to provide the necessary structure to modulate membrane morphology [53]. However, the actin cytoskeleton and its regulatory proteins are not merely structural proteins: they also act as scaffolds for stably positioning and anchoring other scaffolding proteins, ion-channels, neurotransmitter receptors, cell-adhesion proteins, and other macromolecules at the cell surface that contribute to the architectural integrity and function of the postsynaptic density (PSD) [54]. They are also necessary for the trafficking of subcellular structures such as endosomes, and for higher brain functions such as memory formation and extinction [55]. At the PSD, the family of PDZ domain-containing scaffold proteins offers a molecular interface between glutamatergic receptors (GluRs) and the cytoskeleton [56,57]. For example, two proteins that contain PDZ domains, PSD95 and SHANK (SH3 and multiple ankyrin repeat domains), can bind through multiple interactions to NMDA receptors and to the metabotropic glutamate receptor (mGluR) [58,59]. The postsynaptic expression of SHANK was shown to enhance presynaptic function [59], suggesting a key role for the SHANK scaffold in synaptic plasticity.
Another group of proteins that are tethered to membranes by lipid groups attached to their C-termini are the Rab proteins. These proteins belong to the Ras superfamily of small GTPases. They are predominantly present in an active state, whereas the dephosphorylation of GTP by hydrolysis to GDP converts Rab proteins to their inactive form. Rab proteins have been implicated in the facilitation of membrane-associated receptor transport. Some key Rab proteins identified as participating in the internalization and recycling of membrane receptors are Rab 5, Rab 4, and Rab 11. The former is located on the cytoplasmic surfaces of the plasma membrane and is specifically associated with the internalization of clathrincoated pits and with the fusion of early endosomes [60,61]. Rab 4 is generally described to facilitate the rapid recycling of receptors directly from endosomes and targeted back to the plasma membrane [62,63]. Rab 11 is associated with the slow recycling of GPCRs via the perinuclear recycling compartment [64]. Thus, the regulation of the number and the availability of receptors at the plasma membrane strongly depends on Rab proteins.

The Endocannabinoid System
ECs are involved in a plethora of physiological and pathological processes in mammalian cells, having effects on mood, appetite, reproduction, immunity, memory, and pain perception [65]. The EC system comprises cannabinoid receptors (CBRs), endogenous cannabinoid ligands (endocannabinoids, Ecs), and the enzymes involved in their synthesis and degradation. Ecs are lipid molecules, synthesized de novo by phospholipase action after hydrolyzing the lipid precursors from the cellular membrane [66]. The Ecs anandamide (N-arachidonoylethanolamine, (AN)) and 2-arachidonoylglycerol (2-AG) are lipid messenger molecules. AN is an endogenous lipid neurotransmitter derived from the polyunsaturated fatty acid arachidonic acid and a member of the N-acylethanolamines (NAEs) family [67]. It is a partial agonist of cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R), and a full agonist of the vanilloid receptor 1 (VR1), whereas 2-AG acts as a full agonist of CB1R and CB2R. Additionally, ECs can activate other "non-CB" receptors, such as the G-protein-coupled receptor 55 (GPR55) and peroxisome proliferator-activated receptors (PPARs) [68][69][70][71].
CB1Rs are expressed at very high levels in a subset of GABAergic interneurons, such as the cholecystokinin (CCK)-containing basket cells in the forebrain [72], and at lower levels on many glutamatergic terminals throughout the brain [73]. Within neurons, quantitative electron microscopy studies revealed that CB1Rs are found mainly on the pre-terminal axonal segment and less on more proximal axons, dendrites, or the cell soma [74].

The Endocannabinoid System in Synaptic Transmission
In contrast to classical neurotransmitters, ECs are produced as needed [75]. Upon postsynaptic depolarization, for example, after activation of metabotropic glutamate, muscarinic, or dopamine D2 receptors [76][77][78], the synthesis of ECs takes place through Ca 2+ influx and the activation of the Gq-protein, or via phospholipase C and D activation ( Figure 1). However, AN and 2-AG have different synthetic and metabolic pathways [79]. While the former is mainly synthesized from N-acyl-phosphatidylethanolamine (NAPE) by NAPEspecific phospholipase D (NAPE-PLD) and metabolized by fatty acid amidohydrolase (FAAH) [79], the latter is predominantly synthesized from 2-arachidonoyl-containing phospholipids by DAG lipase (DAGL) and metabolized by monoacylglycerol lipase (MAGL). Once synthesized, ECs are released into the synaptic cleft and are able to activate CBRs in presynaptic and/or nearby GABAergic, glutamatergic, and cholinergic neurons [77,[80][81][82][83]. containing phospholipids by DAG lipase (DAGL) and metabolized by monoacylglycerol lipase (MAGL). Once synthesized, ECs are released into the synaptic cleft and are able to activate CBRs in presynaptic and/or nearby GABAergic, glutamatergic, and cholinergic neurons [77,[80][81][82][83]. The latter retrograde EC modulation of GABA terminals enables the depolarizationinduced suppression of inhibition (DSI) through a transient suppression of GABA released onto the postsynaptic neuron, thereby disinhibiting it. EC action on CBRs located on the glutamate terminal, on the other hand, favors a depolarization-induced suppression of excitation (DSE) [84,85] by inhibiting glutamate release, hence activating postsynaptic neurons. However, the role of DSI is reported to predominate over that of DSE due to differences in CB1R sensitivity between inhibitory and excitatory synapses [86]. Importantly, the predominant GABA suppression in the midbrain is suggested to be the The latter retrograde EC modulation of GABA terminals enables the depolarizationinduced suppression of inhibition (DSI) through a transient suppression of GABA released onto the postsynaptic neuron, thereby disinhibiting it. EC action on CBRs located on the glutamate terminal, on the other hand, favors a depolarization-induced suppression of excitation (DSE) [84,85] by inhibiting glutamate release, hence activating postsynaptic neurons. However, the role of DSI is reported to predominate over that of DSE due to differences in CB1R sensitivity between inhibitory and excitatory synapses [86]. Importantly, the predominant GABA suppression in the midbrain is suggested to be the mechanism that induces dopamine (DA) release at mesocorticolimbic and nigrostriatal sites [87] associated with reward-based learning and also with addiction [88,89]. The EC system has a central role in the modulation of synaptic transmission throughout the CNS. ECs regulate behavior through reward learning, by modulating mesolimbic reward circuits that include the ventral tegmental area (VTA), the nucleus accumbens (NAc), and the lateral habenula (LHb). It is these CNS functions that have given ECs notoriety in relation to drug abuse and addiction behaviors. Furthermore, EC-like lipid-signaling molecules are increasingly understood as neuromodulators able to regulate dopaminergic transmission, as well as reward and addiction behavior. Thus, upon interacting with different receptors within the plasma membrane, ECs stimulate a great variety of signaling pathways. As we will see, membrane microdomains are key players in modulating signal transduction through the organization of CBRs.
Other nonspecific mechanisms of action, e.g., involving the alteration of the host lipid bilayer, have been suggested for ECs [90]. AN, for instance, was shown to reduce the ionic current of many voltage-gated ion channels in the presence of antagonists of CB1Rs and CB2Rs [91][92][93][94], thus suggesting a receptor-independent mechanism for the modulation of multiple membrane protein functions.

The Endocannabinoid System Organization in Membrane Microdomains
Once activated by ECs, either CB1R or CB2R trigger a signaling cascade through the inhibitory Gi and Go subtypes of the G proteins (i.e., Gai1, Gai2, Gai3, Gao1, Gao2) [95,96]. Their activation by ECs leads, in turn, to the inhibition of adenylyl cyclase, the activation of mitogen-activated protein kinases, the inhibition of certain voltage-gated calcium channels [97,98], and the activation of G protein-linked inwardly rectifying K+ channels [96,99,100] (Figure 2).
As shown in Figures 1 and 2, the CB1R can also modulate ion channel proteins [96]. The effects of CBR activation are long lasting because of the several secondary messenger molecules involved in processes that play major roles in neuronal plasticity [101,102]. This makes apparent the diversity of signaling pathways that can be elicited by different CB1R and CB2R agonists. However, the great capacity of CBRs to couple to different subtypes of G proteins does not make them less specific in triggering downstream signaling transduction mechanisms. The plasma membrane plays a major role in confining cannabinoid responses, both spatially and temporally. Cholesterol/sphingomyelin-rich domains provide CBRs with dynamic and organized platforms where assembly of signaling complexes can take place and, in addition, prevent crosstalk between different pathways [103]. Reinforcing the relevance of membrane domains for the EC system signal selectivity, CB1R binding and signaling are demonstrated to be influenced by membrane compartmentalization [103].
Chemicals such as methyl-β-cyclodextrin deplete cholesterol from the plasma membrane ( Figure 2) and prevent the onset of cell death by completely blocking the ability of AN to induce superoxide generation, phosphatidylserine exposure, and p38 MAPK activation. However, the use of a CBR antagonist did not prevent cell death in primary hepatic stellate cells [104], thus suggesting a more complex CBR-lipid interface interaction that is disturbed upon cholesterol depletion. In agreement with this observation, the activation of CB1Rdependent adenylate cyclase signaling by AN is reported to be almost doubled by methylβ-cyclodextrin treatment, pointing to the relevance of these lipid platforms in cell signaling processes [103]. Conversely, upon membrane cholesterol enrichment, CB1R-dependent signaling was shown to be reduced by half in primary cultures and immortalized cell lines [105,106].
CB1Rs are physically associated with ordered lipid domains in a cholesterol-dependent manner ( Figure 2). Cholesterol depletion alters AN-induced CB1R endocytosis and its subsequent trafficking to the lysosomal compartment, pointing to the importance of lipid platforms in the intracellular trafficking of the CB1R [107]. No such information is available on CB2Rs. Lipid platforms can physically sequester different signaling components, preventing crosstalk between different pathways [108], or favor internalization of CB1Rs via caveolae-related endocytosis (Figure 2), negatively regulating CB1R function [109]. For example, the desensitization process of CB1R involves phosphorylation of specific serine residues in the intracellular loop III and C terminal regions. Phosphorylation of these serine residues is performed by protein kinase C (PKC) [110] and G-protein-coupled receptor kinase 3 (GRK3) [111], promoting the recruitment of β-arrestin 2 [111]. These three molecules (PKC, GRK3, and β-arrestin 2) are either resident at or dependent on Lo lipid domains [112], which may explain why, upon disruption of these domains, the desensitization process of CB1R fails and signaling through the CB1R is sustained. Once phosphorylated by PKC and GRK3, β-arrestin 2 is recruited and CB1R is targeted for internalization towards either low pH endosomes and subsequent recycling back to the cell surface, or to late endosomes and lysosomes for degradation ( Figure 2). The former endocytic and recycling cycle of the CB1R is facilitated by the small GTPases Rab5 and Rab4, respectively ( Figure 2) [62].
naling molecules are increasingly understood as neuromodulators able to regulate dopa-minergic transmission, as well as reward and addiction behavior. Thus, upon interacting with different receptors within the plasma membrane, ECs stimulate a great variety of signaling pathways. As we will see, membrane microdomains are key players in modulating signal transduction through the organization of CBRs.
Other nonspecific mechanisms of action, e.g., involving the alteration of the host lipid bilayer, have been suggested for ECs [90]. AN, for instance, was shown to reduce the ionic current of many voltage-gated ion channels in the presence of antagonists of CB1Rs and CB2Rs [91][92][93][94], thus suggesting a receptor-independent mechanism for the modulation of multiple membrane protein functions.

The Endocannabinoid System Organization in Membrane Microdomains
Once activated by ECs, either CB1R or CB2R trigger a signaling cascade through the inhibitory Gi and Go subtypes of the G proteins (i.e., Gai1, Gai2, Gai3, Gao1, Gao2) [95,96]. Their activation by ECs leads, in turn, to the inhibition of adenylyl cyclase, the activation of mitogen-activated protein kinases, the inhibition of certain voltage-gated calcium channels [97,98], and the activation of G protein-linked inwardly rectifying K+ channels [96,99,100] (Figure 2).  induces Gi/o-dependent inhibition of adenylyl cyclase (AC) activity and activation of different MAPK cascades. CB1R positively regulates inwardly rectifying K + channels, whereas it negatively regulates voltage-gated Ca 2+ channels. In addition, the CB1R can activate PLC at the plasma membrane and can also signal through non-G proteins such as the adaptor protein FAN. The phosphorylated CB1R is a target of β-arrestin. The attenuation of CB1R signaling at cholesterol-rich lipid domains occurs through a Rab5-dependent endocytic mechanism. Once internalized, the CB1R may either be recycled back to the plasma membrane through a Rab4-dependent mechanism or further targeted to lysosomal degradation. The CB2R does not reside in or interact with Lo lipid platforms. Alteration of ordered lipid domain integrity either by depleting or supplying cholesterol is shown to inhibit or enhance the internalization of CB1R, respectively. The inset depicts a closer view of the cholesterol-rich Lo lipid domain (marked with a dotted line in the main figure). Cholesterol molecule in gray, sphingolipids in red and other phospholipids in yellow.
Although CB1R is present at the plasma membrane, in HEK-293 cells approximately 85% of the heterologously expressed receptors are localized in intracellular vesicles [62], suggesting a predominantly intracellular localization at a steady state. In agreement with these observations, the intracellular localization of CB1R was previously described in AtT20 cells [113] and hippocampal neurons [114]. Intracellularly located CB1Rs cannot interact with ECs or exogenous cannabinoids, thus preventing stimulation. These data support CB1Rs being constitutively endocytosed. Lo lipid domain instability associated with the diminution of cholesterol content at the plasma membrane would lead to a reduction of the endocytic process and an increase in the number of CB1Rs at the plasma membrane available for ligand interaction. This explains why cholesterol depletion from plasma membranes can favor an enhanced cellular response to ECs, by augmenting the availability of such receptors.
CB2R is also regulated by the Rab family. Specifically, Rab 5 has been implicated, as is also the case with CB1R, in the modulation of the endocytic internalization, whereas Rab11 is reported to participate in a slower recycling process via the perinuclear recycling compartment [64].
Agonist-CBR interactions also mediate lipid raft dynamics. If CB1R are activated in a transient manner, sphingomyelin breakdown is initiated, and ceramides accumulate through functional coupling with the adaptor protein FAN [115]. Conversely, if CB1R are continuously stimulated, G protein-dependent de novo ceramide synthesis takes place through activation of serine palmitoyl transferase activity [116].

Endocannabinoid Interactions with Endocannabinoid Receptors within the Plasma Membrane
The plasma membrane plays a major role in the interaction between AN and CBRs. In order to deliver its biological message, after being synthesized, AN leaves the postsynaptic membrane, crosses the synaptic cleft, and finds its way to CB1Rs at the presynapse. AN is an amphipathic derivative of arachidonic acid with a polar head of an ethanolamine group. It is considered a lipid-derived neurotransmitter molecule with enhanced water solubility. Because of its lipidic nature, however, it is very unlikely that AN interacts with CB1Rs via their extracellular region; the plasma membrane on the other hand provides an adequate microenvironment for AN to interact with the transmembrane helices of the receptor [117,118]. For this to occur, AN has to firstly penetrate the lipid bilayer and, secondly, diffuse in the plasmalemma to find its binding site [117,119]. Cholesterol again is a central player in the process of guiding AN towards its receptor. Cholesterol by itself suffices to act as the AN transporter at the plasma membrane [120]. AN is deemed to exhibit a specificity for cholesterol in biological membranes over other lipids [121]. This interaction is thought to occur through a hydrogen bond between the -OH group of the sterol and the -NH group of AN [121] that, once established, enables cholesterol to trigger the insertion of AN to the plasma membrane through a flip-flop mechanism favoring the development of van der Waals interactions that stabilize the complex [121]. It should be noted that this process is unlikely to occur within Lo lipid domains, where cholesterol is tightly packed with sphingomyelin [122,123] and is hence inaccessible to AN; it is more likely to occur in non-raft, liquid-disordered domains of the plasma membrane with greater accessibility to AN. Since CB1Rs in the brain are mostly found in Lo lipid domains [107,124], to reach its binding site, AN must penetrate these domains either by passive diffusion (owing to its lipid nature) or by a membrane transport system. Again, cholesterol seems to be the molecule that performs these transport and delivery functions.
From an energetic point of view, AN has a much higher affinity for CB1R than for cholesterol (energy of interaction~−136 kJ mol −1 vs. −30.3 kJ mol −1 ) [125]. This thermodynamic characteristic has led to the suggestion that, when the AN-cholesterol complex reaches the CB1R, AN detaches from cholesterol and moves towards the receptor. The AN binding site on the CB1R has been localized between the transmembrane 6 (TMH6) and 7 (TMH7) regions of the GPCR [121]. TMH7 of the CB1R carries a CARC domain [23,121] that is available for cholesterol interaction. It is proposed that cholesterol could attract AN back, contributing to the release of AN from the binding site, and enabling the CB1R to revert to the unbound state after receptor activation. In addition, the cholesterol-AN complex leaves the polar head of the neurotransmitter fully accessible for enzymatic hydrolysis by FAAH [121]. All in all, the plasma membrane provides the EC system with multiple compartments to regulate the synthesis, transport, and even degradation of its neurotransmitters. It harbors metabolic enzymes and further contributes to the enhancement or abolishment of signal transduction.

nAChR
nAChRs are excitatory, cationic pLGICs that reside in presynaptic, postsynaptic, and extrasynaptic membranes in the central and peripheral nervous system [126][127][128][129][130]. They are essential players in neurotransmission across different types of synapses and in muscle contraction. nAChRs can be activated by their natural neurotransmitter, ACh), or by a wide variety of ligands [131,132]. nAChRs at the plasma membrane can adopt multiple conformational states. Upon agonist exposure, the closed state of the channel becomes activated and in the "open state", the influx of small cations can take place. After depolarization of the cell, the channel can adopt a desensitized state that is unable to be activated by ligand binding or return to a closed state.
Seventeen different subunits (α1-α10, β1-β4, γ, ε, and δ) encoded by seventeen genes in vertebrates that combine and form either homo-or hetero-pentameric structures, have been described [133]. The muscle-type nAChR found at the neuromuscular junction is formed from four distinct subunit types organized in an (α) 2 βγδ pentamer, the fetal γsubunit being replaced by the ε-subunit in the adult. The subunit composition of the nAChR determines the kinetics of the conformational stages of the channel. For example, while α4β2 nAChRs desensitize rapidly when exposed to the agonist nicotine, α7 nAChRs do not desensitize as fast [134][135][136]. Also, the subunit composition affects the selective cationic permeability of the nAChR and the pharmacological affinities of various agonists [137][138][139][140]. nAChRs influence synaptic plasticity by increasing intracellular Ca 2+ release, inducing long-term potentiation (LTP) and favoring a depolarization state. The activation of presynaptically located nAChRs results in the release of many neurotransmitters including dopamine, norepinephrine, GABA, and Glu in a Ca 2+ -dependent manner. Heteromeric α4β2 and homomeric α7 nAChRs are the most abundant neuronal subtypes [141,142]. The activation of these receptors in hippocampal interneurons indirectly affects neurotransmitters release (Glu or GABA) by activating voltage gated calcium channels [143,144]. Furthermore, presynaptic α4β2 nAChR activation by the agonist nicotine is reported to induce dendritic spine enlargement as a consequence of increased Glu concentration and of glutamatergic neurotransmission [145], thus affecting synaptic plasticity. Activation of other neuronal nAChRs, such as the α3β4 nAChR, at presynaptic sites is shown to stimulate tetrodotoxin-insensitive GABA release via T-type voltage gated calcium channels and Ca 2+ from internal stores [146].
At postsynaptic sites, the activation of nAChRs also produces significant inward (depolarizing) currents in neurons in many brain regions and modulates synaptic plasticity. A7 nAChR at postsynaptic sites can regulate glutamate receptors and, hence, glutamatergic signaling [147,148], as well as GABAergic interneuron activity [149,150]. Halff and coworkers proposed a mechanism by which α7 nAChRs would modulate synaptic potentiation independently of fast excitatory transmission [7]. They report that the activation of α7 nAChRs at postsynaptic sites recruits GluA1 receptors from the surface pool of mobile extrasynaptic receptors, this in turn contributing to stabilization of GluA1 receptors in the neuronal spine and producing an increase in the density of this subtype of glutamatergic receptors. However, the participation of functional PSD-95 scaffold protein family members is required to anchor GluA1 macromolecules at the postsynaptic membrane. Both activation and inhibition of α7 nAChRs in the prelimbic cortex result in the induction of LTP [151]. Modulating network excitability by cholinergic signaling must therefore be finely regulated for adequate neural excitability and plasticity to take place.

nAChR Localization within the Plasma Membrane
The neuronal nAChRs are found in brain areas considered to be involved in learning, cognition, and memory, such as the basal forebrain, hippocampus, cerebellum, and the temporal cortex [152]. nAChRs are found within lipid platforms in the plasma membrane. These cholesterol/sphingolipid-rich lipid domains determine nAChR nanocluster topography function and mobility on cell surfaces [153,154]. Numerous papers over the past 40 years have provided data on the multiplicity of effects of cholesterol on the peripheral nAChRs found in skeletal muscle, and in the electromotor synapse of electric fish.
nAChRs are influenced by cholesterol concentration at various levels of organization, within multiple time windows, and during ontogenetic development and adulthood [20,155]. Cholesterol homeostasis dysregulation produces alterations in the biophysical properties of the membrane bilayer that affect the mobility and protein-protein interactions of neurotransmitter receptors [22].
Cholesterol/sphingolipid/ceramide-rich lipid platforms are required for both muscletype and neuronal nAChR trafficking to the plasma membrane [153,[156][157][158][159]. Indeed, our group has provided evidence that disruption of these lipid platforms leads to both altered nAChR function and cell-surface expression [153,154].
Lowering cholesterol levels at the plasma membrane causes rapid nAChR (muscle type) internalization, and compensatory gain-of-function of the nAChRs remaining at the plasma membrane [160][161][162]. Studies using stimulated emission depletion (STED) [163] and single-molecule localization (SMLM) [164,165] superresolution microscopies further show contrasting changes in the distribution of nAChR nanoclusters and individual molecules upon cholesterol depletion or enrichment. In addition, changes in the cholesterol concentration of the membrane followed using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy [153] and SMLM [164,165] have also been shown to modify the translational mobility of the receptor in the plane of the plasma membrane. Likewise, cholesterol content in the cell-surface membrane modulates neuronal nAChR subtypes. In a recent study by Báez-Pagán and coworkers, a reduction in the macroscopic response of the neuronal α7 nAChR subtype was described as cholesterol to phospholipid ratios increased [166]. The α7 nAChR subtype of nAChRs is predominantly located in cholesterol-rich Lo domains [157,159,167,168]. Other studies from our laboratory indicate that long-term inhibition of cholesterol biosynthesis can differentially augment cell-surface levels of both α4β2 and α7 nAChRs in the neurites and soma of rat hippocampal neurons [169].
Bearing in mind that neuronal nAChRs can be found on both the soma and synaptic terminals of excitatory and inhibitory neurons in different brain regions (Figure 1), the lipid microenvironment of the plasma membrane where they reside plays a crucial role in the modulation of nAChR responses. Additionally, alteration of cholesterol content at the plasma membrane will affect neuronal excitability through the modulation of the nAChR channels and by indirectly impacting on GABAergic and glutamatergic transmission, in turn possibly impinging on multivariable physiological or pathophysiological outcomes.

nAChR and EC Receptor Crosstalk
The overlapping distribution of CBRs and nAChRs is not fortuitous. Their distribution anatomically overlaps in brain areas such as the midbrain, the hippocampus, and the amygdala. Many papers support the notion that nicotinic and EC systems interact bidirectionally in the brain reward pathway [170][171][172][173][174][175][176][177][178][179][180][181][182][183][184][185][186][187]. Buczynski and coworkers, reported that nicotine self-administration in rats modified AN levels in the VTA [188]. Furthermore, fluctuations in AN but not 2-AG are associated with withdrawal from nicotine [189]. The lipophilic nature of ECs allows their incorporation into the plasma membrane, and their presence is shown to modulate the action of alcohol and volatile anesthetics on α7 nAChRs [190,191], suggesting that an alteration of the physicochemical properties of the plasma membrane is taking place. Using Xenopus oocytes, Oz and coworkers demonstrated that AN can reversibly inhibit nicotine-induced currents in a concentration-dependent manner without significantly affecting its half maximal effective concentration (EC50) value. These authors suggest that AN behaves as a noncompetitive antagonist of α7 nAChRs [192,193]. Likewise, AN was shown to inhibit the peak amplitudes of α4β2 nAChR-mediated currents in SH-EP1 cells [194] and in myenteric neurons [92,93]. In addition, synthetic cannabinoids have been reported to modulate cholinergic neurotransmission in the hippocampus [170,[185][186][187]. In the latter, nAChRs and CB1Rs play a role in the cognition-impairing effects of the main psychoactive ingredient of cannabis, δ-9-tetrahydrocannabinol (THC) [195]. In the mesolimbic dopamine system, they are implicated in brain reward processes [195][196][197]. The blockade of α7 nAChR by methyllycaconitine at non-toxic concentrations reduces the behavioral and neurochemical effects of THC related to its abuse [198]. It has therefore been suggested that drugs that block α7 nAChR could be useful agents in the treatment of cannabis abuse in humans [198]. Contrarily, THC is reported to increase the response of the α7 nAChR to ACh by 128% [199]. Cannabidiol (CBD), the other most abundant component of cannabis, is reported to reduce cigarette consumption by 40% and to reduce the response of the α7 nAChR to ACh by 49% [199]. Understanding the complex interactions between the cholinergic and the endocannabinoid systems in the nervous system is critical to increasing our chances of using cannabinoids in the clinic as therapeutic tools.
Cohen and coworkers reported that SR141716 (rimonabant), a CB1R antagonist, blocks the motivational and dopamine-releasing effects of nicotine in rats [200], while other authors have also observed that rimonabant decreases the motivation to self-administer nicotine [201]. In support of the existence of a physiological interaction between the cannabinoid and cholinergic systems, genetic deletion of CB1R in knockout mice was demonstrated to inhibit nicotine-induced rewarding effects, evaluated by a conditioned place preference paradigm (CPP) [202,203]. In addition, a blockade of the CB1R in various areas of the brain, such as in the shell of the NAc, the basolateral amygdala, the prelimbic cortex, and in the bed nucleus of the stria terminalis, was reported to produce a reduction in nicotine-seeking behavior [204,205]. Genetic deletion or pharmacological inhibition of FAAH is shown to enhance the expression of nicotine CPP [203], thus suggesting that ECs play an important role in the rewarding properties of nicotine. Furthermore, CB1R stimulation in rats was reported to increase the motivation to self-administer nicotine and to enhance cue-induced reinstatement of nicotine-seeking behavior [206]. In contrast, neither the activation nor the inhibition of CB2R was shown to produce effects on the motivation to obtain nicotine or nicotine intake in rats [207]. However, studies performed in mice have documented the relevance of CB2R receptors on the rewarding/reinforcing properties of nicotine [208,209]. Thus, the current literature supports a distinct profile of CB1R and CB2R effects on behavior, and suggests that there may be important species differences mediating these effects.
The modulatory crossover effects taking place between CBRs and nAChRs at the plasma membrane point to EC and cholinergic systems as holding significant promise for the development of novel therapeutic strategies in behavior and addiction.

Concluding Remarks
Biological membranes are much more complex than double-layered lipid barriers acting simply as a boundary between the intracellular content and the extracellular medium. They are highly organized and multifaceted structures that provide an asymmetrical interface where many transport and enzymatic processes can simultaneously occur. The direct observation of nanoscopic lateral heterogeneities in the plane of the membrane accomplished in the last two decades has provided structural grounds that help explain how these dynamic platforms control and govern signal transduction, neurotransmitter synthesis, metabolism, and degradation. Here, we briefly reviewed the importance of these supramolecular structures and mechanisms using the CB1R and the nAChR as an example of two interacting cell-surface signaling systems. We also stressed the importance of the neutral lipid cholesterol in the phenomena involving these paradigmatic receptors. Cholesterol plays a direct role in orchestrating the membrane organization and endocytosis of the two receptor systems, contributes to the transport of the lipidic endocannabinoid AN to and from the CB1R, and participates in the regulation of the number, distribution, and functional states of nAChRs.
Author Contributions: F.J.B. and A.S.V. conceived the work, searched the literature, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding:
No funding is associated with this review.


s2fieldsofstudy: Biology Medicine

text:
Gallic Acid Ameliorates the Inflammatory State of Periodontal Ligament Stem Cells and Promotes Pro-Osteodifferentiation Capabilities of Inflammatory Stem Cell-Derived Exosomes

The slow proliferation rate and poor osteodifferentiation ability of inflammatory periodontal membrane stem cells extracted from periodontitis tissues (i-PDLSCs) account for poor efficiency in treating inflammatory bone loss. Exosomes reportedly have inducible and relatively stable components, allowing them to promote inflammatory bone repair, but obtaining i-PDLSCs exosomes with the ability to promote osteodifferentiation is challenging. In the present study, i-PDLSCs were extracted from periodontal membrane tissues of patients with severe periodontitis, and in vitro induction with gallic acid (GA) significantly promoted the proliferative activity of i-PDLSCs at a concentration of 10 mM, with TC0 of 11.057 mM and TC50 of 67.56 mM for i-PDLSCs. After mRNA sequencing, we found that GA could alleviate oxidative stress in i-PDLSCs and increase its mitochondrial membrane potential and glucose aerobic metabolism level, thus promoting the osteodifferentiation of i-PDLSCs. After exosomes of i-PDLSCs after GA induction (i-EXO-GA) were isolated by differential centrifugation, we found that 200 ug/mL of i-EXO-GA could remarkably promote the osteodifferentiation of i-PDLSCs. Overall, our results suggest that GA induction can enhance the proliferation and osteodifferentiation in primary cultures of i-PDLSCs in vitro, mediated by alleviating oxidative stress and glycometabolism levels in cells, which further influences the osteodifferentiation-promoting ability of i-EXO-GA. Overall, we provide a viable cell and exosome induction culture method for treating inflammatory alveolar defects associated with periodontitis.

Introduction
Periodontitis is a chronic inflammatory disease associated with bacterial biofilm and characterized by the destruction of the tooth-supporting tissues, which can eventually lead to loose teeth or even edentulism [1]. In the pathogenesis of periodontitis, multiple inflammatory factors are activated [2], resulting in pathological resorption of alveolar bone, known as alveolar inflammatory bone loss [3]. Furthermore, bone loss results in insufficient bone volume in the alveolar bone, significantly limiting the feasibility of dental implantation. It has been established that periodontitis can affect the efficacy of guided bone regeneration (GBR) treatment [4].
Over the years, periodontal ligament stem cells (PDLSCs) have been widely applied as seed cells for tissue-engineered repair of bone deficiencies and alveolar bone defects in studies on alveolar bone tissue engineering and regeneration [5]. Contemporarily, it has been shown to promote periodontal tissue regeneration with good osteodifferentiation potential. However, the extraction of therapeutically available PDLSCs in periodontitis is unpractical since the extracted cells mostly exhibit an inflammatory phenotype with poor proliferative activity and osteodifferentiation potential [6]. Another paradox is that PDLSCs transferred into the periodontitis environment can be regulated by inflammatory factors and eventually fail to promote alveolar bone healing. In recent years, it has been proposed that exosomes (EXO) secreted by PDLSCs are bioactive components in the construction of cell-free tissue engineering scaffolds [7]. Exosomes of PDLSCs are extracellular vesicles with a diameter of 30-150 nm, rich in proteins, miRNA, RNA, lipids, and DNA. Current evidence suggests that transplanted PDLSCs promote tissue regeneration mainly through paracrine action. Exosomes play a key role in stem cell therapy as a major component of the stem cell secretion system [8,9]. Therefore, PDLSCs exosomes can replace PDLSCs during therapy and compensate for their limitations, such as long primary culture time and intolerance to long-term storage. Interestingly, exosomes exhibit a relatively stable composition after being secreted and are not subject to changes in the inflammatory environment [10]. Therefore, exosomes from PDLSCs have huge prospects for treating inflammatory bone loss associated with periodontitis. Nevertheless, it remains unclear how efficient exosomes of PDLSCs can be obtained from periodontitis tissues.
We hypothesized that the culture of inflammatory PDLSCs with improved inflammatory phenotype and osteodifferentiation could facilitate the acquisition of exosomes that promote osteodifferentiation. It has been established that the Chinese medicine, Xipayi mouth rinse, made from gallic, is effective against oral bacteria that cause simple gingivitis and mild periodontitis [11]. Moreover, it can prevent and treat oral bacterial infectious diseases and reduce plaque and gingival bleeding. Gallic acid (GA) is a polyphenolic organic compound reported to have various biological functions such as anti-inflammatory, anti-tumor, and antioxidant damage [12]. GA can alleviate periodontitis by inhibiting nuclear factor (NF-κB) upregulation and phosphorylation induced by lipopolysaccharide (LPS). It inhibits the excessive release of inflammatory cytokines IL-1β and TNF-α and reduces bone destruction, while GC may reduce LPS-induced osteoclast activation and alveolar bone resorption by upregulating the OPG/s-RANKL pathway to achieve therapeutic effects [13,14]. However, the effect of GCon cellular function and exosomes of inflammatory PDLSCs is unknown. In our study, we implemented an array of experiments to assess the osteodifferentiation promoting ability of GA-induced i-PDLSCs exosomes (i-EXO-GA) in vitro, which may provide a new strategy for the treatment of inflammatory bone loss caused by periodontitis.

Primary Cell Extraction and Culture of PDLSCs
The present study was approved by the Ethics Committee of Guangdong Second Hospital of Traditional Chinese Medicine. Primary cell extraction was conducted after obtaining informed consent from the patients. Healthy periodontal ligament stem cells (h-PDLSCs) were harvested from the periodontal ligament tissues of healthy teeth extracted before orthodontic treatment of 3 patients. Inflammatory periodontal ligament stem cells (i-PDLSCs) were harvested from the inflammatory periodontal ligament tissues of 3 patients with stage III periodontitis (alveolar bone loss extending to mid-third of root and beyond, tooth loss due to periodontitis of ≤4 teeth, probing depth ≥6 mm, and vertical bone loss ≥3 mm). PDLSCs were not harvested from patients with diabetes, metabolic diseases, recent hormone use, tumors, etc. During primary cell extraction and cell culture, 1/3 of the periodontal ligament tissue in the root was scraped and washed with DPBS solution containing BI 1-antibody and 3% gentamicin for 1 min. The periodontal ligament tissue was added into DPBS containing 3 mg/mL Collagenase I and digested in a water bath at 37 • C for 1 h. After digestion, the cells were filtered through a 70 µm filter membrane to obtain the cell suspension. After centrifugation at 100× g for 5 min, the cells were resuspended and precipitated with a complete medium (DMEM medium containing 10% exosome-free serum and 1% penicillin-streptomycin). The cell density was adjusted to 1 × 10 4 cells/mL. The cells were inoculated in a T25 cell culture flask and placed in a 5% CO 2 incubator at 37 • C. After 24 h, the non-adherent cells were removed by completely changing the liquid, and the liquid was completely changed once in the following 2-3 d. The cells were subcultured when the cell density reached 80%.

GA Induction Culture
The second generation of i-PDLSCs was seeded in 6-well plates, and the complete medium containing different concentrations of GA was added for culture induction, which was named i-PDLSCs-GA.

MTT Cytotoxicity Assay
The second generation of i-PDLSCs was inoculated in 96-well plates and induced with different concentrations of GA for 24 h. h-PDLSCs incubated in a complete culture medium were used as the control group. The MTT assay was used to detect the cytotoxicity of different concentrations of GA on i-PDLSCs. GA was dissolved in DPBS solution and diluted to 400, 200, 100, 50, 25, 10, 5, 2.5, 1.25, and 1 mM in a complete culture medium to induce i-PDLSCs. According to the kit instructions, 20 µL MTT working solution was added to each well. After 4 h of culture and centrifugation at 2000× g for 10 min, 200 µL DMSO was added to each well and fully oscillated. The absorbance value was read at 490 nm using a microplate reader. Cell survival rate (%) = (i-PDLSCs-GA group average OD value/h-PDLSCs group average OD value) × 100%. The maximum toxic concentration (TC0) and half toxic concentration (TC50) were calculated by the probit regression function of SPSS (version 24.0) software (IBM, Armonk, NY, USA).

mRNA Sequencing
RNA was extracted from h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA cultured for 3 d, and mRNA sequencing was performed. Total RNA was extracted by the TRIZOL method. Cells were passaged into a T75 cell culture flask, and 5 mL TRIZOL was added. An amount of 4 mL was frozen in liquid nitrogen for mRNA sequencing. A total of 250 µL chloroform was added to 1 mL solution and shaken until a pink color was observed, kept at room temperature for 10 min, and centrifuged at 12,000× g for 5 min at 4 • C. The supernatant was taken into the EP tube and added to 500 µL isopropanol; the mixture was shaken and allowed to stand at room temperature for 15 min. An amount of 1 mL of 75% ethanol was added, and the precipitate was washed and centrifuged at 4 • C for 10 min at 12,000× g. The supernatant was removed, and 50 µL DEPC water was added to dissolve RNA after being dried in the ventilation cabinet. An amount of 1 µL was taken for Nanodrop detection. An OD value of 1.8-2.0 was acceptable, and the remaining samples were stored at −80 • C for further use. The purity and integrity of total RNA were analyzed by Agilent 2100 BioAnalyzer system, and samples with RNA integrity number ≥8.0 were selected for further detection. A lysing reagent was added to lyse the mRNA into short fragments at moderate temperature in a thermostatic mixer. A one-stranded cDNA was synthesized using the interrupted mRNA as a template, and a two-stranded synthesis reaction system was formulated to synthesize two-stranded cDNA. After purification and recovery, we conducted sticky end repair and addition of base "A" to the 3' end of cDNA with ligated connectors, followed by fragment size selection and PCR amplification for library construction according to the kit instructions. The constructed libraries were quality-checked by Agilent 2100 Bioanalyzer and Qubit and sequenced using Illumina sequencers. The data obtained from Illumina HiSeqTM 2000 sequencing, called Raw reads or Raw data, were then subjected to quality control (QC) to determine if the sequencing data is suitable for subsequent analysis. After the above QC, a series of follow-up analyses such as gene and transcript quantification, various analyses based on gene expression levels (principal component, correlation, condition-specific expression, differential gene screening, etc.), exon quantification, gene structure optimization, variable splicing, new transcript prediction and annotation, SNP detection, Indel analysis, gene fusion, etc. were performed. The differentially expressed genes (DEGs) among the screened samples were analyzed by GO functional significance enrichment analysis, pathway significance enrichment analysis, clustering, protein interaction network, and transcriptional factors for more in-depth mining.

Quantification of DEG-Related Proteins by ELISA
DEGs were screened according to the mRNA sequencing results, and the protein expression levels corresponding to the DEGs were verified by ELISA. The h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA were cultured for 3 d, and SOD1, SOD2, CAT, SIRT1, and SIRT3 levels were determined according to the kit's instructions.

Detection of Mitochondrial Membrane Potential
The JC-1 mitochondrial membrane potential kit was used to detect h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA cultured for 3 days. The cells in each group were seeded in a confocal 6-well plate. JC-1 staining was conducted according to the kit's instructions and added to the plate. The cells were incubated at 37 • C for 15 min. The negative control group was set as h-PDLSCs-CCCP with 10 µM CCCP. An inverted laser confocal microscope was used to stimulate and photograph at 488 and 594 nm. It has been established that JC-1 exists in the mitochondrial matrix as a polymer when the mitochondrial membrane potential is high, and the polymer fluorescence can be measured at 594 nm. However, JC-1 exists as a monomer when the mitochondrial membrane potential is low and cannot be aggregated in the mitochondrial matrix. JC-1 is mostly dissociated in the cytoplasm, and the monomer fluorescence can be measured at 488 nm. Fluorescence images were quantified by the ratio of two fluorescence channels (594/488 nm).

Detection of Oxidative Stress Level
MDA and SOD levels were detected in h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA cultured for 3 days using ELISA kits.

Detection of Glycometabolism Level
The metabolites of h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA cultured for 3 d were determined by ATP and lactic acid detection kits. The activity of the glycolysis rate-limiting enzyme was determined by 6-phosphate fructose kinase (PFK-1) activity and pyruvate kinase (PK) activity detection kit. The expression levels of citrate synthase (CS) and malate dehydrogenase (IDH), NADH dehydrogenase 1-β3 (NDUFB3), and succinate dehydrogenase complex β subunit (SDHB) were detected by RT-qPCR. The reaction system included SsoFast Eva Green Supermix 5 µL, upstream primer (10 µM) 0.5 µL, downstream primer (10 µM) 0.5 µL, cDNA template 2 µL, and ultrapure water 2 µL. The reference gene β-tubulin was used for data normalization. The expression of related genes was calculated by the 2 −∆∆CT method. The primer sequences are listed in Table 1. Alkaline phosphatase (ALP) staining was performed on h-PDLSCs, i-PDLSCs, i-PDLSCs-GA, and h-PDLSCs-CCCP with 10 µM CCCP cultured for 7 d. The cells in each group were inoculated on a 12-well plate for 7 d. The plate cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. The color buffer, BCIP solution, and NBT solution were prepared into a working solution according to 300:1:2. An amount of 500 µL working solution was added to each well and incubated at room temperature for 30 min. The cells were washed with distilled water and photographed with a micro camera.
The h-PDLSCs, i-PDLSCs, i-PDLSCs-GA, and h-PDLSCs-CCCP cultured for 14 d were stained with alizarin red. The cells in each group were inoculated in 12-well plates for 14 d. The cells in the well plates were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and added with 1 mL alizarin red staining solution. After static staining at room temperature for 30 min, the cells were washed with distilled water and photographed with a micro-camera.

Extraction of Exosomes by Differential Centrifugation
h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA cells cultured for 3 d were inoculated into 10 T75 culture flasks. After the cells were cultured to 80% confluence, they were washed twice with PBS. The complete medium containing 10% exosome-free serum and 1% double antibody was used for total exchange. The medium in each flask was 50 mL. After 72 h of culture, the supernatant of the cell culture medium (500 mL in each group) was collected for exosome extraction. The supernatant was centrifuged at 300× g for 10 min at 4 • C to transfer the supernatant and then centrifuged at 10,000× g for 10 min at 4 • C to remove dead cells and cell debris. The supernatant was carefully collected and transferred to a 50 mL open-ended ultracentrifugal tube for 4 • C and 100,000× g for 70 min, and the supernatant was discarded. The precipitate was suspended with 2 mL DPBS and then transferred to a 12.5 mL tube for centrifugation at 4 • C for 70 min at 100,000× g. Each centrifuge tube was suspended with 200 µL PBS. After filtering with a 0.22 µm pore size, it was loaded with an EP tube and stored at −80 • C to avoid repeated freezing and thawing. Exosomes extracted from the supernatants of h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA cell culture medium were recorded as h-EXO, i-EXO, and i-EXO-GA, respectively.

Exosomes Identification
(1) Determination of BCA protein concentration: An amount of 20 µL exosome solution from each group was taken for reaction with the BCA working solution and incubated at 37 • C for 30 min. The OD values of the sample group and the standard group at 562 nm were read by the microplate reader, the standard curve was plotted, and the protein concentration of the sample was calculated. The samples were adjusted to the same protein concentration for subsequent detection. (2) Identification of exosome markers by Western blot: A total of 100 µL exosome extracts from each group were mixed with 20 µL protein sample buffer and heated at 95 • C for 5 min. The protein separated by SDS polyacrylamide gel electrophoresis was transferred to PVDF membranes incubated in 5% skim milk powder (prepared with PBS solution) at room temperature for 1 h. They were then incubated with a 1:1000 diluted primary antibody (Anti-CD81, Anti-CD9, Anti-ALIX, Anti-β-actin) overnight at 4 • C, followed by a 1:5000 dilution horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. ECL luminescent solution was added and incubated for 3 min for chemiluminescence imaging. (3) Particle size analysis: An amount of 1 mL exosome samples was slowly injected into the Nanosight nanoparticle size analyzer, and the thermometer probe was placed in the copper hole. After adjusting to the appropriate focal length, the particle size and concentration were determined.

Coculturing of i-PDLSCs and Exosomes
h-EXO and i-EXO exosomes were adjusted to 300 µg/mL in a complete culture medium, and i-EXO-GA exosomes were adjusted to gradient concentrations of 100, 200, and 300 µg/mL. After digestion, i-PDLSCs were resuspended in an exosome-containing medium and inoculated in 12-well plates.

Detection of Osteodifferentiation Index
Alkaline phosphatase (ALP) staining: ALP staining was performed on the cocultured cells of each group at 7 d, using the same method as in Section 2.2.9; (2) Alizarin red staining: The cells cocultured in each group were stained with alizarin red on the pore plate at 14 d; (3) RT-PCR: RT-qPCR was used to detect the expression levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and type I collagen (COL-I) in the cocultured cells of each group at 0, 7, 14, and 21 d. The 12-well plate cells were transferred to the EP tube, frozen and crushed into powder in liquid nitrogen, and the total RNA was extracted by RNA kit. The Prime ScriptTM RT Master Mix reverse transcription kit was used for reverse transcription, and the primers were added to the real-time fluorescence quantitative PCR analyzer. The measured data were used to calculate the expression of related genes by the 2 −∆∆CT method. The primer sequences used are listed in Table 2. Graphpad prism 9.3 software was used for statistical analysis. The data were expressed as x ± s. One-way analysis of variance was used to compare multiple groups of samples. A p-value < 0.05 was statistically significant.

GA Promotes the Proliferation Activity of i-PDLSCs
The MTT assay was conducted on i-PDLSCs induced by different concentrations of GA ( Figure 1). GA induction promoted the proliferation of i-PDLSCs, especially with 10 mM GA. In contrast, 25 mM GA induced a relatively weak stimulatory effect on cell proliferation. We found that the maximum non-toxic concentration (TC0) and the half toxic concentration (TC50) of GA on i-PDLSCs were 11.057 and 67.56 mM, respectively ( Figure 1A). As shown in Figure 1B, the proliferation activity of i-PDLSCs was lower than that of h-PDLSCs, and the cell survival rate was 66.87% of h-PDLSCs. When the GA concentration was 5 and 10 mM, i-PDLSCs-GA exhibited higher proliferation activity than h-PDLSCs. However, when the GA concentration was above 25 mM, i-PDLSCs-GA exhibited lower proliferation than h-PDLSCs, indicating that high concentrations of GA were cytotoxic. Accordingly, 10 mM GA was selected for culture induction in subsequent experiments. The micrographs of i-PDLSCs induced by different concentrations of GA ( Figure 1C) showed that cell proliferation was not significantly different from the uninduced group when the GA concentration was 10 mM and 11.057 mM, and the cell proliferation was significantly inhibited when the GA concentration was 67.56 mM.

mRNA Sequencing Showed That the GA-Induced Culture Improved Oxidative Stress and Glycometabolism of i-PDLSCs
The mRNA sequencing results of h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA on day 3 of GA induction are shown in Figure 2. A total of 1145 genes were detected by sequencing, and 74 DEGs exhibited a two-fold difference in expression between both groups ( Figure  2A). BMP2B1, COL2A1, SOD2, SIRT3, CAT, and RAB27A exhibited high expression in h-PDLSCs. Moreover, IL1A, TNSF11, TNFSF2, MMP13, MEK, and DNM1 were highly expressed in i-PDLSCs. Finally, ALP, SIRT1, AKAP9, PFDN5, and ITGB5 were highly expressed in i-PDLSCs-GA. Gene ontology (GO) analysis ( Figure 2B) of DEGs was conducted in terms of biological process, cellular component, and molecular function. The DEGs were significantly enriched in biological processes, including hypoxia, oxidative stress, aerobic respiration, osteoblast differentiation, and acute inflammation. Moreover, the DEGs were significantly enriched in cell components, including extracellular regions,

mRNA Sequencing Showed That the GA-Induced Culture Improved Oxidative Stress and Glycometabolism of i-PDLSCs
The mRNA sequencing results of h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA on day 3 of GA induction are shown in Figure 2. A total of 1145 genes were detected by sequencing, and 74 DEGs exhibited a two-fold difference in expression between both groups ( Figure 2A). BMP2B1, COL2A1, SOD2, SIRT3, CAT, and RAB27A exhibited high expression in h-PDLSCs. Moreover, IL1A, TNSF11, TNFSF2, MMP13, MEK, and DNM1 were highly expressed in i-PDLSCs. Finally, ALP, SIRT1, AKAP9, PFDN5, and ITGB5 were highly expressed in i-PDLSCs-GA. Gene ontology (GO) analysis ( Figure 2B) of DEGs was conducted in terms of biological process, cellular component, and molecular function. The DEGs were significantly enriched in biological processes, including hypoxia, oxidative stress, aerobic respiration, osteoblast differentiation, and acute inflammation. Moreover, the DEGs were significantly enriched in cell components, including extracellular regions, cytoplasm, mitochondria, and nucleus. The molecular function of differentially expressed genes was mainly related to peroxidase activity, deacetylase activity, and superoxide dismutase activity. KEGG pathway enrichment analysis was used to describe the signaling pathways involved in differentially expressed genes ( Figure 2C). The main signaling pathways involved include glucose/energy metabolism, anti-oxidative stress, TGF-β signaling pathway, NAD metabolism, etc. The expression levels of SOD1, SOD2, CAT, SIRT1, and SIRT3 in each group were verified by ELISA ( Figure 2D). The results showed no significant difference in SOD1 protein expression among the three groups. The protein expression level in h-PDLSCs and i-PDLSCs-GA was significantly higher than in i-PDLSCs (p < 0.05). The sequencing results showed that high expression of inflammation-related genes in i-PDLSCs after GA treatment could cause changes in cellular oxidative stress, glycometabolism, osteodifferentiation, etc.

GA Treatment Alleviates Oxidative Stress in i-PDLSCs
On day 3 of GA induction, the mitochondrial membrane potential, malondialdehyde (MDA), and superoxide dismutase (SOD) of h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA were analyzed ( Figure 3). As seen in Figure 3A(i), during mitochondrial membrane potential detection, the JC-1 probe could not enter the mitochondria at low mitochondrial membrane potential, showing green fluorescence JC-1 monomer. When the mitochondrial

GA Treatment Alleviates Oxidative Stress in i-PDLSCs
On day 3 of GA induction, the mitochondrial membrane potential, malondialdehyde (MDA), and superoxide dismutase (SOD) of h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA were analyzed ( Figure 3). As seen in Figure 3A(i), during mitochondrial membrane potential detection, the JC-1 probe could not enter the mitochondria at low mitochondrial membrane potential, showing green fluorescence JC-1 monomer. When the mitochondrial membrane potential was high, the JC-1 probe aggregated in the mitochondrial matrix in the form of polymers, showing red fluorescence. The results showed that the i-PDLSCs group and the h-PDLSCs + CCCP group treated with mitochondrial depolarization inducer CCCP had higher green fluorescence intensity and lower mitochondrial membrane potential. In comparison, the red fluorescence intensity of h-PDLSCs and i-PDLSCs-GA increased significantly. As shown in Figure 3A(ii), quantitative analysis of fluorescence ratio showed that the mitochondrial membrane potential of i-PDLSCs-GA cells induced by GA was significantly higher than i-PDLSCs and h-PDLSCs + CCCP (p < 0.05). During the quantification of oxidative stress indexes ( Figure 3B), ELISA of oxidative stress marker MDA and anti-oxidative stress marker SOD showed that MDA and SOD of i-PDLSCs-GA were significantly different from i-PDLSCs (p < 0.05), and their SOD indexes were significantly higher than h-PDLSCs (p < 0.05). The above results suggested that the mitochondrial membrane potential of i-PDLSCs was low, and the oxidative stress level was high. After GA induction culture, the mitochondrial membrane potential and oxidative stress levels were significantly improved. MDA and anti-oxidative stress marker SOD showed that MDA and SOD of i-PDLSCs-GA were significantly different from i-PDLSCs (p < 0.05), and their SOD indexes were significantly higher than h-PDLSCs (p < 0.05). The above results suggested that the mitochondrial membrane potential of i-PDLSCs was low, and the oxidative stress level was high. After GA induction culture, the mitochondrial membrane potential and oxidative stress levels were significantly improved.

Enhancement of Glycometabolism in i-PDLSCs Induced by GA
On day 3 of culture induction with GA, the glycometabolism products, glycolytic enzyme activity, tricarboxylic acid cycle enzyme mRNA levels, and oxidized phosphorylase complex subunit mRNA levels in h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA were analyzed ( Figure 4). As shown in Figure 4A, the ATP production in i-PDLSCs was lower than

Enhancement of Glycometabolism in i-PDLSCs Induced by GA
On day 3 of culture induction with GA, the glycometabolism products, glycolytic enzyme activity, tricarboxylic acid cycle enzyme mRNA levels, and oxidized phosphorylase complex subunit mRNA levels in h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA were analyzed ( Figure 4). As shown in Figure 4A, the ATP production in i-PDLSCs was lower than in h-PDLSCs, and ATP production in i-PDLSCs-GA increased within 72 h, reaching a level similar to h-PDLSCs at 48 h. On the contrary, the lactic acid content in i-PDLSCs-GA decreased within 72 h. The activities of glycolytic rate-limiting enzymes 6-phosphofructose kinase (PFK-1) and pyruvate kinase (PK) are shown in Figure 4B. The enzyme activities in i-PDLSCs-GA decreased within 72 h, reaching a level similar to h-PDLSCs at 48 h. As shown in Figure 4C, the mRNA expression of citrate synthase (CS) and malic dehydrogenase (IDH), the key enzymes of the tricarboxylic acid cycle, were detected by RT-qPCR. The results showed that the expression of CS and IDH was upregulated in the GA-induced culture. Results in h-PDLSCs and i-PDLSCs-GA were significantly different from i-PDLSCs after 24 h (p < 0.05). As shown in Figure 4D, RT-qPCR was used to detect the mRNA expression of NADH dehydrogenase 1-β3 (NDUFB3) and succinate dehydrogenase complex β subunit (SDHB). NDUFB-3 expression in h-PDLSCs and i-PDLSCs-GA was significantly different than in i-PDLSCs after 24 h (p < 0.05). SDHB levels in i-PDLSCs-GA were significantly higher than in i-PDLSCs after 48 h (p < 0.05). The above results showed that the aerobic glucose metabolism in i-PDLSCs was significantly increased after GA induction for 24 h. i-PDLSCs-GA decreased within 72 h, reaching a level similar to h-PDLSCs at 48 h. As shown in Figure 4C, the mRNA expression of citrate synthase (CS) and malic dehydrogenase (IDH), the key enzymes of the tricarboxylic acid cycle, were detected by RT-qPCR. The results showed that the expression of CS and IDH was upregulated in the GA-induced culture. Results in h-PDLSCs and i-PDLSCs-GA were significantly different from i-PDLSCs after 24 h (p < 0.05). As shown in Figure 4D, RT-qPCR was used to detect the mRNA expression of NADH dehydrogenase 1-β3 (NDUFB3) and succinate dehydrogenase complex β subunit (SDHB). NDUFB-3 expression in h-PDLSCs and i-PDLSCs-GA was significantly different than in i-PDLSCs after 24 h (p < 0.05). SDHB levels in i-PDLSCs-GA were significantly higher than in i-PDLSCs after 48 h (p < 0.05). The above results showed that the aerobic glucose metabolism in i-PDLSCs was significantly increased after GA induction for 24 h.

Effect of Oxidative Stress and Aerobic Glucose Metabolism on Osteodifferentiation of i-PDLSCs
The results of cellular alkaline phosphatase (ALP) staining on day 7 of GA induction, alizarin red staining, and osteodifferentiation-related gene expression level assay on day 14 are shown in Figure 5. We found that h-PDLSCs and i-PDLSCs-GA exhibited higher ALP levels while i-PDLSCs had lower ALP levels ( Figure 5A). Accordingly, almost no positive ALP staining was observed when h-PDLSCs-CCCP were treated with a sublethal dose (10 μM) of CPPP, a mitochondrial depolarization inducer. As shown in Figure 5B, alizarin red staining showed a similar trend, with many positively stained mineralized nodules in h-PDLSCs and i-PDLSCs-GA, few mineralized nodules in i-PDLSCs, and the lowest level of mineralized nodules in h-PDLSCs-CCCP. As seen in Figure 5C, there were no statistical differences in the expression of RUNX2 in the h-PDLSCs and i-PDLSCs groups on days 0, 7, 14, and 21, while the OCN and COL-I levels were lower in the i-PDLSCs group on days 14 and 21, and the h-PDLSCs-CCCP group had the lowest expression. On days 14 and 21, the expression of each osteodifferentiation-related gene was significantly higher in the i-PDLSCs-GA group, showing a more significant contribution to osteodifferentiation. It can be seen that alleviated mitochondria-related oxidative stress

Effect of Oxidative Stress and Aerobic Glucose Metabolism on Osteodifferentiation of i-PDLSCs
The results of cellular alkaline phosphatase (ALP) staining on day 7 of GA induction, alizarin red staining, and osteodifferentiation-related gene expression level assay on day 14 are shown in Figure 5. We found that h-PDLSCs and i-PDLSCs-GA exhibited higher ALP levels while i-PDLSCs had lower ALP levels ( Figure 5A). Accordingly, almost no positive ALP staining was observed when h-PDLSCs-CCCP were treated with a sublethal dose (10 µM) of CPPP, a mitochondrial depolarization inducer. As shown in Figure 5B, alizarin red staining showed a similar trend, with many positively stained mineralized nodules in h-PDLSCs and i-PDLSCs-GA, few mineralized nodules in i-PDLSCs, and the lowest level of mineralized nodules in h-PDLSCs-CCCP. As seen in Figure 5C, there were no statistical differences in the expression of RUNX2 in the h-PDLSCs and i-PDLSCs groups on days 0, 7, 14, and 21, while the OCN and COL-I levels were lower in the i-PDLSCs group on days 14 and 21, and the h-PDLSCs-CCCP group had the lowest expression. On days 14 and 21, the expression of each osteodifferentiation-related gene was significantly higher in the i-PDLSCs-GA group, showing a more significant contribution to osteodifferentiation. It can be seen that alleviated mitochondria-related oxidative stress and enhanced aerobic glucose metabolism in the GA induction culture promoted the osteodifferentiation of i-PDLSCs-GA, while the loss of membrane potential of mitochondria after CCCP treatment significantly reduced the level of differentiation of h-PDLSCs, which exhibited good osteodifferentiation.

Identification of PDLSCs Exosomes
Exosomes were extracted from the culture supernatants of h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA cells by differential centrifugation and named h-EXO, i-EXO, and i-EXO-GA, respectively ( Figure 6). The BCA protein assay was used to detect the content of exosomes in the supernatant of cell culture medium per unit volume. The results showed no significant difference between h-EXO and i-EXO-GA ( Figure 6A) exosomes isolated from the supernatant of the cell culture medium, while levels in i-EXO were significantly less than the two groups (21.38 ± 2.01, 21.69 ± 0.80 and 12.33 ± 2.37 μg/mL, respectively). The surface markers CD81, CD9, and ALIX were detected, and β-actin was not detected in exosome samples by Western blot (Figure 6B, supplementary materials). The NTA particle size analysis results of exosomes are shown in Figure 6C. It can be seen that the particle sizes of the three groups of exosomes ranged from 30 to 150 nm, consistent with the particle size range of exosomes. However, i-EXO contained fewer particles than the other two groups. The above results substantiated that exosomes were successfully isolated from

Identification of PDLSCs Exosomes
Exosomes were extracted from the culture supernatants of h-PDLSCs, i-PDLSCs, and i-PDLSCs-GA cells by differential centrifugation and named h-EXO, i-EXO, and i-EXO-GA, respectively ( Figure 6). The BCA protein assay was used to detect the content of exosomes in the supernatant of cell culture medium per unit volume. The results showed no significant difference between h-EXO and i-EXO-GA ( Figure 6A) exosomes isolated from the supernatant of the cell culture medium, while levels in i-EXO were significantly less than the two groups (21.38 ± 2.01, 21.69 ± 0.80 and 12.33 ± 2.37 µg/mL, respectively). The surface markers CD81, CD9, and ALIX were detected, and β-actin was not detected in exosome samples by Western blot (Figure 6B, supplementary materials). The NTA particle size analysis results of exosomes are shown in Figure 6C. It can be seen that the particle sizes of the three groups of exosomes ranged from 30 to 150 nm, consistent with the particle size range of exosomes. However, i-EXO contained fewer particles than the other two groups. The above results substantiated that exosomes were successfully isolated from the supernatant of the cell culture medium.

The Function of i-EXO-GA in Promoting Osteodifferentiation and Concentration Screening
The concentrations of h-EXO and i-EXO were adjusted to 300 μg/mL, and the concentration gradients of i-EXO-GA were set to 100, 200, and 300 μg/mL. Coculture with i-PDLSCs was carried out in vitro, and the osteodifferentiation-related genes were quantified ( Figure 6). The ALP staining results on day 7 are shown in Figure 6D. A high concentration of i-EXO induced a disproportionate decrease in ALP staining of i-PDLSCs cells. The high concentration of h-EXO could induce ALP expression, but the staining intensity was lower than in the i-EXO-GA group. i-EXO-GA concentration was positively correlated with ALP staining intensity. Alizarin red staining results on day 14 were consistent with ALP staining, as shown in Figure 6E. The quantification of osteodifferentiation-related gene expression at days 0, 7, 14, and 21 are shown in Figure 6F. We found that the expression of RUNX2 at each time point was comparable between h-EXO and i-EXO groups. On days 7 and 14 of OCN, and day 21 of the COL-I group, h-EXO showed a more

The Function of i-EXO-GA in Promoting Osteodifferentiation and Concentration Screening
The concentrations of h-EXO and i-EXO were adjusted to 300 µg/mL, and the concentration gradients of i-EXO-GA were set to 100, 200, and 300 µg/mL. Coculture with i-PDLSCs was carried out in vitro, and the osteodifferentiation-related genes were quantified ( Figure 6). The ALP staining results on day 7 are shown in Figure 6D. A high concentration of i-EXO induced a disproportionate decrease in ALP staining of i-PDLSCs cells. The high concentration of h-EXO could induce ALP expression, but the staining intensity was lower than in the i-EXO-GA group. i-EXO-GA concentration was positively correlated with ALP staining intensity. Alizarin red staining results on day 14 were consistent with ALP staining, as shown in Figure 6E. The quantification of osteodifferentiation-related gene expression at days 0, 7, 14, and 21 are shown in Figure 6F. We found that the expression of RUNX2 at each time point was comparable between h-EXO and i-EXO groups. On days 7 and 14 of OCN, and day 21 of the COL-I group, h-EXO showed a more obvious osteodifferentiation-promoting ability than i-EXO, indicating that h-EXO yielded a definite osteodifferentiation-promoting effect on i-PDLSCs. However, on days 14 and 21, different concentrations of i-EXO-GA yielded a higher osteodifferentiation promoting effect than the h-EXO and i-EXO groups. During the comparison of different concentrations of i-EXO-GA, the osteodifferentiation function of low concentration i-EXO-GA was worse than medium and high concentration groups at some time points. The difference between the medium and the high concentration groups was not significant, except for COL-I on day 14. Overall, our study revealed that 200 µg/mL i-EXO-GA could promote osteodifferentiation.

Discussion
One major tissue engineering application of PDLSCs is alveolar bone regeneration induction [15]. Current evidence suggests that PDLSCs can self-renew and have multidirectional differentiation potential, which are basic characteristics of mesenchymal stem cells [16]. Thus, osteogenic-induced PDLSCs are widely regarded as seed cells for alveolar bone tissue engineering. Although the osteodifferentiation function of PDLSCs is wellestablished [5], the repair of bone defects during clinical application involves a more complex pathological environment. Periodontitis is widely acknowledged as an important underlying disease that leads to alveolar bone defect and insufficient bone mass [17]. Periodontitis can negatively impact the primary culture of autologous PDLSCs and induce a decline in osteodifferentiation [18]. A study compared PDLSCs derived from normal periodontal tissue and inflammatory periodontal tissues and found that i-PDLSCs showed a weaker osteodifferentiation but better proliferation ability [19]. In our study, the MTT assay found that the proliferation activity of i-PDLSCs was significantly lower than h-PDLSCs. Accordingly, severe periodontitis tissues were selected for primary culture in our study. i-PDLSCs may be subject to damage induced by severe inflammation, leading to a low proliferation activity. Overwhelming evidence substantiates that during periodontitis, PDLSCs can stimulate macrophages to secrete inflammatory factor IL-1β by activating its endoplasmic reticulum stress, thereby mediating bone resorption and inhibiting bone tissue regeneration [20]. We previously established that exosomes of inflammatory PDLSCs were rich in RANKL and TNF-α, which could promote the osteodifferentiation of macrophages, possibly via exosome-mediated intercellular communication prompted pathological bone resorption in periodontitis [21]. In this study, we found that i-PDLSCs had higher expression levels of TNF-α and IL-1β by mRNA sequencing, consistent with cytokine expression in the above studies.
During the MTT assay, when 10 mM GA was used for induction of PDLSCs, similar findings were obtained to previous studies that 1 mM has no cytotoxicity on Vero cells, 4.17 mM has no cytotoxicity on lymphocytes, and 31.76 mM is the half lethal dose of HeLa cells [22,23]. This study demonstrated cytotoxicity on i-PDLSCs cells with GA concentrations above 11.057 mM. It has been established that GA has anti-inflammatory, antioxidant, anti-tumor, anti-platelet aggregation, and bacteriostatic effects [24]. In oral diseases, GA plays an antibacterial role in changing the morphology and structure of dental plaque biofilm, reducing the density of biofilm and killing bacteria in biofilm, and inhibiting the secretion of TNF-α [25]. Thus, it plays an anti-inflammatory role. Therefore, the xipayi mouth rinse, with gallic acid as the main component, is used as a gargle for the clinical treatment of periodontitis [26]. In this study, transcriptome sequencing showed that h-PDLSCs expressed many genes related to osteodifferentiation, and i-PDLSCs cells highly expressed TNF-α and other inflammatory factors, consistent with the function and phenotype reported in previous studies. i-PDLSCs-GA exhibited high expression of genes that promote antioxidant stress, glucose aerobic metabolism, and osteodifferentiation.
Previous studies found that GA could inhibit the activation of apoptosis-related proteins, reduce the level of ROS in mitochondria, and downregulate the expression of COX-2 and iNOS [13], thereby reducing the concentration of lipopolysaccharide in vivo [27]. It is wellestablished that inflammation can cause oxidative stress. This study found that induction culture with GA can improve the oxidative stress of PDSLCs-i-GA and promote aerobic glucose metabolism, proving that GA has a prominent effect on improving the proliferative activity and inflammatory phenotype of PDLSCs.
During analysis of oxidative stress and glycolysis levels in PDSLCs, we found that mitochondria in i-PDLSCs possess a low membrane potential, high oxidative stress, and high glycolysis levels. Current evidence suggests that phagocytosis of neutrophils activated by pathogenic microbial invasion can increase oxygen consumption in periodontitis tissue, and the reperfusion period after hypoxia can lead to increased ROS production in periodontal cells [28]. Hence, following damage induced by the inflammatory response, i-PDLSCs mainly exhibit glycolysis energy consumption and high oxidative stress levels [29]. In contrast, the oxidative stress levels of i-PDLSCs-GA were alleviated after GA induction treatment with downregulated MDA and upregulated SOD levels [30], and the glycometabolism mode gradually changed from glycolysis to aerobic oxidation, comparable to h-PDLSCs [31]. However, little is currently known about glycometabolism during osteodifferentiation. It is widely thought that cells prefer energy produced via glycolysis which is more effective during osteodifferentiation [32,33]. Meanwhile, our findings suggest that i-PDLSCs adopt glycolysis after the mitochondrial membrane potential is decreased and mitochondrial function is impaired by oxidative stress injury. Glycolysis is a prerequisite for the tricarboxylic acid cycle and oxidative phosphorylation. After GA induction, ATP production is increased while the lactate content and activity of glycolytic rate-limiting enzymes are decreased, and i-PDLSCs-GA produce more pyruvate as a substrate for the tricarboxylic acid cycle through glycolysis, instead of lactate as a glycolytic product. Albeit PDLSCs have promising osteodifferentiation potential, the osteodifferentiation potential of i-PDLSCs extracted from periodontitis tissues was not significant. To the best of our knowledge, no study has hitherto investigated the effect of GA on osteodifferentiation of cells. Our transcriptome sequencing results revealed that i-PDLSCs-GA exhibited high expression levels of SOD2 (MnSOD), SIRT3, and other genes, suggesting that the effect of GA on the osteodifferentiation ability of PDLSCs may be mediated through the MnSOD/SIRT3 signaling pathway, which reportedly promotes osteodifferentiation by protecting cells from oxidative stress damage and promoting aerobic metabolism [32].
Exosomes are tiny vesicles containing nucleic acids, proteins, lipids, etc. secreted by cells [34]. Exosomes are well-established to express specific markers, such as CD9, CD63, CD81, ALIX, etc. [35,36]. In the present study, exosome samples with the same protein concentration were used to measure the markers mentioned above but not β-actin, which is consistent with the established characteristics of exosomes. It has been shown that the exosomes of MSCs mediate paracrine/autocrine effects after being taken up by target cells [37], promote the synthesis of growth factors, cytokines, regulatory factors, signal peptides [38], and other bioactive molecules by target cells, and activate the migration of stem cells to the site of tissue damage to participate in repair, as well as regulate the directional differentiation and specific molecular expression of stem cells [39,40]. Current evidence suggests that exosomes contain less immunogenic material than stem cells and enable target and drug delivery adjustment [41]. More importantly, exosomes can be extracted and frozen for storage, allowing easy adjustment of drug delivery concentrations [42,43]. When treating alveolar bone defects associated with periodontitis, stem cells may undergo alterations in cellular functions in the inflammatory microenvironment in vivo, whereas exosomes contain relatively stable components [44]. Thus, exosomes have huge potential for clinical application. Recent studies have also focused on inflammatory bone loss in the alveolar bone associated with periodontitis, using normal PDLSCs-derived exosomes with biomaterials for treatment [45]. This study found that exosomes of normal PDLSCs can inhibit excessive activation of Wnt and promote the osteodifferentiation ability of inflammatory PDLSCs [43]. Using normal PDLSCs exosomes with matrix gelma/β-tricalcium phosphate composites can result in cell-free therapy for bone loss caused by periodontitis [46,47]. In this study, we concluded that although h-EXO has a good osteodifferentiation ability, periodontitis is the most common underlying disease in clinical patients with alveolar bone defects, which makes it difficult to provide h-PDLSCs, while the primary-acquired ones are mostly i-PDLSCs with an inflammatory phenotype. For the primary culture of inflammatory tissue cells, different commercial media containing growth factors such as FGF are available but expensive, while in vitro induction culture of GA is relatively cheaper, yielding i-EXO-GA with good osteodifferentiation ability, suggesting it has huge prospects for application in the treatment of alveolar bone defects associated with periodontitis.
Indeed, further exploration of the expression of miRNA and protein components in i-EXO-GA is warranted, given that the mechanism of its contribution to osteodifferentiation remains unclear. Moreover, the effectiveness of i-EXO-GA treatment of alveolar bone defects associated with periodontitis with biomaterials warrants further investigation in animal studies.

Conclusions
In this study, we substantiated that GA could improve the proliferative activity and oxidative stress level of i-PDLSCs, increase aerobic metabolism in cells, and improve the function of its exosome to contribute to osteodifferentiation. This study provides a theoretical basis and technical method for the primary culture of PDLSCs and induction culture before exosome acquisition. Accordingly, i-EXO-GA has huge prospects for application in treating alveolar bone defects associated with periodontitis.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/life12091392/s1, Table S1: Lane information of western blot. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data used to support the findings of this study are included in the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.